The laser-induced breakdown spectroscopy (LIBS) technique is a very powerful method for determining the elemental composition of the surface of a material. This composition is obtained by measuring the emission lines coming from the atoms or the ions constituting a transient plasma induced at the surface of the material by a laser beam, and the elemental analysis of this surface can be obtained by scanning said surface by means of a laser source performing successive shots according to a regular grid, so as to map the surface of this material. At each laser shot, the emission spectrum of the chemical elements being investigated is recorded, as are the coordinates for focusing of the beam at the surface of the material. It is then possible to reconstitute a point-by-point image of the surface of the material at a selected wavelength corresponding to a given chemical element. If a calibration method is applied, the variations in intensity of the image obtained by mapping indicate the abundance of this element at the surface of the material.
However, a major drawback of this technique is that the very high number necessary for carrying out a high-resolution mapping of the surface (resolution of between 300 nm and 1 μm) implies analysis times of several hours or even several days, as soon as the surface area analyzed exceeds a few square millimeters. This slowness of the image acquisition makes LIBS inapplicable as a method of analysis for a large number of applications in biology, and more particularly for genomics and proteomics.
In the genomics field, biochips represent a major revolution in the molecular biology techniques of the past ten years. By enabling the simultaneous study of the level of expression of several hundred, or even several thousand, genes, they make it possible to grasp the impact of a disease or of a stress (e.g. resulting from radiation, from pollution or from taking a medicament) at the level of the complete genome of an individual. These techniques are thus becoming increasingly used in modern biology.
Biochips are divided up into two major families, comprising microfluidic chips and probe-array chips. The latter are organized in arrays of “spots” or measuring points, and they are generally obtained by depositing or synthesizing, at precise coordinates on a passive support, molecular probes formed from biopolymers such as DNA, proteins or antibodies, for example. These probe-array biochips make it possible to identify the targets present in a biological sample when these targets hybridize specifically at each “spot” of probes.
There are, on the one hand, high-complexity biochips (more than 5000 spots) for pan-genomic studies and, on the other hand, low-complexity and medium-complexity biochips, which are dedicated to a given purpose (e.g. therapeutic tests, biological detector).
The current probe-array biochip technology has a certain number of major limitations, in particular:                the high steric hindrance of fluorescent labels, which sporadically modify the recognition between probes and targets and thus leads to numerous measurement artefacts, decreasing the reproducibility of the experiments;        the absence of quantitative measurement, which prohibits comparison of the levels of expression between two different targets; and        the high cost of this current technology, both in terms of production and in terms of implementation.        
It is the reason why several alternatives to this technology have recently been developed, with in particular:                “RT-PCR” technologies in microfluidic cards (“Reverse Transcriptase Polymerase Chain Reaction”, i.e. chain amplification by polymerase after reverse transcription of a ribonucleic acid into complementary DNA), which make it possible to amplify up to 386 different targets in parallel, with simplification of the implementation and improved detection, which are, however, penalized by the absence of quantitative measurement for a real comparison between targets, by the limitation of the number of targets to be analyzed (much lower than a low-complexity biochip) and by a high implementation cost;        biochips on “Nylon” film, based on the hybridization of targets in a large volume and chemiluminescence labeling, which also provide simplified implementation and improved detection, but which are nevertheless penalized by the absence of quantitative measurement, the large reaction volume required (which is limiting for analyzing samples of low concentration) and the very high production and implementation costs; and        new concepts of biochips without labeling, which are based on the detection of the target by measuring impedence or by surface plasmon resonance (SPR), and which have in particular been described in David F et al., Bioscience Bioelectron. 2005, in Li C. M. et al., Front Biosci. 2005 or in Macanovic A. et al., Nucleic Acid Research 2004, but which do not make it possible to quantify the number of targets, pose a problem for preparing high-density chips and involve, both for the impedence measurement technique and for the SPR technique, measurement artefacts due to the varying sizes and conformations of the targets.        
ICP (inductively coupled plasma) spectrometry methods have also been developed, cf. Inchul Yang et al., Analytical Biochemistry (2004), vol. 335, 150-161 or Heinrich F. Arlinghaus et al., Analytical Chemistry (1997), vol. 69, No. 18, 3747-3753, said methods making it possible to assay the phosphorus contained in a nucleic acid in order to estimate, for example, the degree of hybridization thereof to a PNA (peptide nucleic acid) biochip.
However, it appears that the use of mass spectrometry for assaying phosphorus using a plasma generated at the surface of a biochip is a slow method (typically taking several hours per cm2 on the biochip), and that the instrumentation necessary for implementing it is expensive. In addition, it should be noted that this ICP spectrometry technique is not quantitative, since it provides only the crude quantity of nucleotides hybridized, without being able to differentiate between the size and the number of the biomolecules.
Patent document US-A-2006/0105354 presents a method of real-time quantification of multiple targets formed from labeled nucleic acids and which are bound to the surface of a probe-array biochip, comprising, in particular, emission of an excitation laser beam at the surface of the array and measurement of the light emission from the hybridized targets in response to this excitation beam.
A major drawback of this method is that it also is not quantitative within the meaning indicated above, and it requires, in addition, the presence of labeled molecules bound to the target molecules.
In the proteomics field, the problem which arises is even more complicated. This is because, in addition to identifying, in a cell extract, the proteins present and also their concentrations, it is necessary to identify their post-translational modifications. This is because the activity of a protein is very often determined by its post-translational modifications. Among the possible modifications, phosphorylations are without doubt the most biologically significant.
Two-dimensional (2D) electrophoresis techniques are the parallel analysis techniques most commonly used for analyzing mixtures of proteins. These techniques consist in migrating a mixture of proteins in a gel, successively in two orthogonal directions, as a function of different physicochemical criteria (e.g. chromatography or electrophoresis). The proteins are then separated according to their affinity with respect to a solvent, their electric charge, their mass, their shape, their modification, etc., and, after having been pigmented with an optionally fluorescent pigment, the proteins are identified according to their position or spot on the 2D gels relative to migrations obtained in a reference gel. To demonstrate the post-translational modifications, in particular phosphorylations, it is necessary to analyze each spot of the gel. Various methods of analysis are possible, such as mass spectrometry, NMR, immunological labeling or the ICP technique, for example.
The demonstration of post-translational modifications is for the moment very laborious to carry out.
For a few years, antibody biochips have been available on the market for analyzing protein extracts. These biochips make it possible to analyze, in parallel, the expression of several hundred genes in their protein form, provided that antibodies specific for the desired proteins exist. However, this technology is limited by the need to label the proteins. Unfortunately, the chromophores used for the labeling randomly modify the affinity of the proteins for their antibody. Furthermore, since the labeling is also random, the results are difficult to interpret and difficult to compare between various experiments. In order to evaluate, by this technique, for example, the degree of post-translational modifications of a given protein between two conditions or two cell types, it is necessary to have two antibodies which exclusively recognize each of the forms of the protein. In addition to being expensive, this is technically very difficult to obtain.
The LIBS technique represents an alternative for the analysis, without prior labeling, of biomolecules segregated on all types of supports (e.g. membrane, gel, plastic support, for example Kapton® support, silicon support), these supports being the main types used in biological analyses. The method for analyzing a biological product by LIBS consists in assaying a chemical element constituting the target biomolecule previously isolated, segregated or purified on a chromatography support, an electrophoresis support, a membrane or a biochip.
As indicated above, the main technological bottleneck of an analysis by LIBS is the slowness of the method, which greatly limits the use of this technique in biology. This is because analysis supports in biology often have a surface area of several square centimeters. The imaging, at a resolution of 10 microns for example, of a 1 cm2 analysis support thus requires 1 000 000 contiguous laser shots to be carried out, at a rate of one shot per position. Of course, this number has to be multiplied by the number of shots to be carried out per position, if the molecule to be analyzed is in the body of the support, as occurs for electrophoresis gels, for example.
In order to carry out this analysis in 1 second, it is necessary to have a mega Hertz UV laser with sufficient energy to induce a plasma on the support at each shot (energy typically greater than 60 microjoules per pulse). In order to carry out this analysis in 1 minute, it is necessary to have a UV laser with a frequency equal to 600 kilo Hertz and sufficient energy to induce a plasma on the support at each shot (also greater than 60 microjoules per pulse). This problem can be minimized by carrying out noncontiguous shots, in return for a degradation in resolution. However, the frequency remains high, even for a very degraded image.
It is possible to use lasers with longer wavelengths, for example around 532 nm, owing to the fact that these lasers generally have higher frequencies and higher energies. However, when moving away from the far UV (266 nm), the energy required to obtain an analyzable plasma increases and, for example, lasers at 532 nm—of sufficient energy—have frequencies in the vicinity of far UV lasers, which then poses the same problem as for the far UV range.